JP5109113B2 - IgE scavenger and antiallergic pharmaceutical composition, cosmetic composition, food composition, beverage composition and feed composition - Google Patents

Description

  The present invention relates to an IgE scavenger and an antiallergic pharmaceutical composition, cosmetic composition, food composition, beverage composition and feed composition.

  Immunoglobulin E (IgE) plays an important role in type I hypersensitivity reactions (anaphylaxis). Usually, an antibody response to an antigen begins with the production of immunoglobulin M (IgM), followed by very low levels of IgE in healthy individuals, along with the production of other isotypes. However, with a genetic background for allergies, large amounts of IgE may be produced in response to certain antigenic stimuli.

  The produced IgE binds to a high affinity IgE receptor (hereinafter referred to as FcεRI receptor) on the surface of a cell membrane such as a mast cell. Mast cells are widely distributed in various tissues and function as effector cells in IgE-dependent immune responses. IgE binding to the FcεRI receptor can be cross-linked by the presence of multivalent (ie, having multiple epitopes) antigens or allergens. This is due to the production of large amounts of IgE as described above. It occurs easily when IgE binds to the surface of cells and the like in large quantities. In mast cells cross-linked with IgE, the biochemical cascade is activated, and mediators that cause allergic reactions (for example, histamine having a vasodilating action) are released. Therefore, IgE has been recognized as being particularly involved in the early stage allergic inflammatory reaction and exerting its function. However, it was thought that it may have various relations in the later stages. In this regard, it has recently been reported that IgE itself has several biological effects on mast cells in the absence of specific antigenic stimulation. Examples include up-regulation of FcεRI receptor, cell life span, cytokine production, PKC activity, increase in actin filament content, and increase in cell adhesion. Therefore, when considering the effectiveness of anti-IgE therapy, not only the function of IgE to increase reactivity in an antigen-dependent manner, but also the function that can activate mast cells by IgE independently in the absence of antigen, It is necessary to keep in mind.

  In fact, various anti-inflammatory effects by anti-IgE treatment have been reported in patients with allergic asthma and allergic rhinitis (see, for example, Non-Patent Document 1). This is very effective in the treatment of

  By the way, the effectiveness of natural products for various diseases is very interesting. Today, these natural products are marketed in many countries as food, tablets and capsules, and can be said to be the main component of new functional foods.

  Recently, among these natural products, components extracted from rice oil and rice germ oil have attracted much attention. Rice is one of the three major crops with production of over 500 million tons annually in the world and over 10 million tons annually in Japan, but about 10% of rice bran is produced in the milling process. Thus, how effectively this rice bran should be used can be said to be one factor that has led to much research on the extracted components.

  As the extract component, ferulic acid (4-hydroxy-3-methoxycinnamic acid) which is a component of rice bran oil and a component of various plants is well known, and this ferulic acid has anti-inflammatory properties, It has been recognized that it has various effects such as anti-neurotoxicity and antiproliferative properties (see, for example, Patent Documents 1 and 2 and Non-Patent Documents 2 to 4).

In addition, as the extraction component, “γ-oryzanol”, which is a general name (generic name) of ferulic acid ester, is also well known, and has a blood cotesterol concentration lowering action, an anti-aging action on skin, Various effects such as an ultraviolet absorption action and an antioxidant action are recognized (see, for example, Patent Documents 3 to 21 and Non-Patent Document 5). However, there is no report that γ-oryzanol has the ability to suppress allergic diseases.
Japanese National Patent Publication No. 10-510803 Special table 2001-504137 Japanese Patent Publication No. 44-58003 JP-A-53-64208 JP 55-55108 A Japanese Unexamined Patent Publication No. 8-188521 JP 58-150600 A Japanese Unexamined Patent Publication No. 57-149248 JP-A-51-56441 Japanese Patent Laid-Open No. 50-160262 JP 63-14797 A JP-A-61-14796 JP 55-162740 JP-A-62-277326 JP-A-61-106512 JP-A-60-130598 JP 50-66746 A JP-A-61-248407 JP 61-40298 Japanese Unexamined Patent Publication No. 60-248611 JP 61-194022 POOLE, JA et al., J. Allergy Clin. Immunol., 115, S376-85, 2005 FERNANDEZ, MA et al., J. Pharm. Pharmacol., 50, 1183-6, 1998 ONO, K. et al., Biochem. Biophys. Res. Commun., 336, 444-9, 2005 YAN, JJ et al., Br. J. Pharmacol., 133, 89-96, 2001 HOU, YZ et al., Eur. J. Pharmacol., 499, 85-90, 2004

  Therefore, the problem to be solved by the present invention is to provide a novel IgE scavenger useful in anti-IgE therapy. Furthermore, it is providing the novel pharmaceutical composition, cosmetic composition, food composition, drink composition, and feed composition which have antiallergic properties.

  The present inventor has intensively studied to solve the above problems. As a result, it was found that γ-oryzanol extracted from rice oil obtained from rice bran and the like has an excellent IgE capturing ability. Furthermore, it has been found that the γ-oryzanol has excellent antiallergic properties.

  That is, the present invention is as follows.

 (1) An IgE scavenger comprising γ-oryzanol, or a pharmacologically acceptable salt thereof, or a hydrate thereof.

 (2) An antiallergic pharmaceutical composition comprising γ-oryzanol, or a pharmacologically acceptable salt thereof, or a hydrate thereof.

 (3) An antiallergic cosmetic composition comprising γ-oryzanol, or a pharmacologically acceptable salt thereof, or a hydrate thereof.

 (4) An antiallergic food composition comprising γ-oryzanol, or a pharmacologically acceptable salt thereof, or a hydrate thereof.

 (5) An antiallergic beverage composition comprising γ-oryzanol, or a pharmacologically acceptable salt thereof, or a hydrate thereof.

 (6) An antiallergic feed composition comprising γ-oryzanol, or a pharmacologically acceptable salt thereof, or a hydrate thereof.

  In the inventions described in (1) to (6) above, examples of γ-oryzanol include those containing cycloartenyl ferulate.

  According to the present invention, an IgE scavenger can be provided as a novel use of γ-oryzanol, which is a rice oil extract. The present invention is based on a novel mechanism in which γ-oryzanol can inhibit the binding reaction between IgE and the above receptor by binding to IgE before binding to the FcεRI receptor. This is extremely useful because sensitization of IgE, which is a premise of type hypersensitivity reaction, is limited and the degranulation reaction can be effectively suppressed even in the presence of allergen substances. Moreover, since γ-oryzanol as an active ingredient is a component derived from a natural product, the present invention is also useful in that it has few problems such as side effects and is excellent in safety.

  Furthermore, according to the present invention, a novel pharmaceutical composition, cosmetic composition, food composition, beverage composition and feed composition having excellent antiallergic properties can be provided as a novel use of the above-mentioned γ-oryzanol. it can.

Hereinafter, the present invention will be described in detail. However, the scope of the present invention is not limited to these descriptions, and modifications other than the following exemplifications can be made as appropriate without departing from the spirit of the present invention.
1. Summary of the Invention The present inventor has focused on the finding that tranilast has an antiallergic action, and examined the presence or absence of an antiallergic action in γ-oryzanol containing a ferulic acid ester having a common skeleton.

  Specifically, a system that measures the degranulation reaction of rat mast cell line RBL-2H3 sensitized with anti-DNP-IgE by antigen (DNP-HSA) stimulation using β-hexosaminidase release as an index The effects of γ-oryzanols, ferulic acid having an aromatic carboxylic acid skeleton and tranilast were investigated. As the γ-oryzanols, γ-oryzanol extracted from rice bran according to a conventional method and cycloartenyl ferulate (CAF) known as a component of γ-oryzanol were used. When anti-DNP-IgE and various concentrations of γ-oryzanol, CAF, ferulic acid and tranilast are treated with RBL-2H3 cells and then stimulated with DNP-HSA, γ-oryzanol and CAF are significantly desensitized. Granule inhibitory action was observed. Tranilast showed a moderate inhibitory effect only in the case of high concentration treatment, but ferulic acid showed almost no inhibitory action. A passive cutaneous anaphylaxis test (PCA test) using rat dorsal skin was also performed with similar results. In other words, γ-oryzanol has a strong mast cell degranulation inhibitory effect, and it was shown that CAF, and further, the effects of its related ester compounds may contribute greatly.

  Next, the present inventor conducted further detailed studies using CAF in order to elucidate the mechanism of the inhibitory action. Anti-DNP-IgE was treated with various concentrations of CAF in advance, and ELISA was performed using IgE antibody. As a result, the detected amount of IgE decreased as the concentration of CAF was increased. However, SDS-PAGE detected CAF-treated Anti-DNP-IgE and untreated one. This result indicates that IgE is not decomposed by the CAF treatment. In addition, the CAF-treated IgE could be easily recovered by centrifugation, and the anti-DNP-IgE content in the supernatant was significantly reduced.

  The above results indicate that γ-oryzanol binds to IgE (ie, captures IgE), thereby suppressing the binding between FcεRI receptor on the mast cell membrane surface and IgE. As a result, it was shown that the degranulation reaction of mast cells is effectively suppressed. And it was shown that such an effect is reproduced by at least CAF which is one component of γ-oryzanol.

  Allergic patients are known to have high IgE concentrations in blood, and in recent years, allergy treatment methods using IgE antibodies are considered promising. The IgE capturing action of γ-oryzanol found by the present inventor shows the possibility of a new therapeutic strategy using a low molecular weight compound instead of IgE antibody therapy.

  The structural formulas of tranilast (1) and ferulic acid (2) described above are shown below. The structural formula of cycloartenyl ferulate (CAF) is shown in FIG. These compounds are commercially available from Wako Pure Chemical Industries, Ltd. and can be easily obtained.

2. IgE scavenger As described above, the IgE scavenger of the present invention comprises γ-oryzanol, or a pharmacologically acceptable salt thereof, or a hydrate thereof.

  In addition, this invention includes use of (gamma)-oryzanol or its pharmacologically acceptable salt, or those hydrates for manufacture of an IgE capture agent.

  In the present invention, “capturing IgE”, that is, “IgE capturing ability” and “IgE capturing action” means binding to IgE and binding to FcεRI receptor present on the surface of a cell membrane such as a mast cell. In other words, it can be said to isolate IgE from mast cells and the like. Specifically, since IgE binds to the FcεRI receptor on the cell membrane surface in its constant region (Fc portion), in order to capture IgE as described above, it binds to the Fc portion of IgE and binds to the FcεRI receptor. A substance capable of reducing the binding activity is an active ingredient.

(1) Active ingredient γ-Oryzanol is a general term for ferulic acid ester compounds such as triterpene alcohol and various plant sterols. γ-oryzanol can usually be extracted and purified from rice oil obtained from rice (such as rice bran). Specifically, the extraction and purification method described in “BLIGH, EG et al., A rapid method of total lipid extraction and purification., Can. J. Biochem. Physiol., 37, 911-7, 1959”. Can be applied.

  As a component contained in γ-oryzanol, for example, cycloartenyl ferulate (CAF), 24-methylenecycloartanyl ferrate, beta-sitosteryl ferrate, campesteryl ferrate and the like can be mentioned. These may be contained alone or in combination of two or more, and are not limited. In general, γ-oryzanol is composed of a plurality of components including CAF and other ferulic acid esters. Among these components, at least CAF is particularly preferable because it has a high IgE supplementation effect alone, and CAF-related active components such as pharmacologically acceptable salts or hydrates of CAF (described later) (unidentified) Are also preferable as well as CAF. As will be described later, the hydrophobicity of CAF is important for its action. Therefore, as the above CAF-related active ingredient, CAF is modified within a range where the hydrophobic region of CAF can be preserved or within a range where the hydrophobicity of CAF can be preserved, and a CAF derivative having IgE supplementing activity is produced. You can also.

  When CAF is contained in γ-oryzanol, the content ratio is limited as long as the ratio of the active substance in the entire γ-oryzanol exists in a certain degree of purity and sufficiently exhibits IgE capturing action. Is not to be done.

  The pharmacologically acceptable salt of γ-oryzanol (hereinafter referred to as the salt of γ-oryzanol) is not limited, and includes a pharmacologically acceptable acid addition salt or basic salt. Examples of acid addition salts include salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid; acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid And salts with organic acids such as succinic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid. Examples of basic salts include salts with inorganic bases such as sodium, potassium, calcium, ammonium, magnesium and aluminum; salts with organic bases such as caffeine, piperidine, trimethylamine, pyridine, methylamine, ethylamine and ethanolamine. And salts with basic amino acids such as lysine, arginine, ornithine and the like.

  The salt of γ-oryzanol can be prepared, for example, by a method using an appropriate acid such as hydrochloric acid or an appropriate base such as sodium hydroxide. Specifically, it can be prepared by treatment using generally known standard protocols in water or in a liquid containing an inert water-miscible organic solvent such as methanol, ethanol or dioxane. .

  When the IgE scavenger of the present invention contains a salt of γ-oryzanol as an active ingredient, the salt may be only one or two or more, and is not limited.

  The above-mentioned γ-oryzanol and salts thereof include, for example, γ-oryzanol and salts thereof that undergo metabolism such as oxidation, reduction, hydrolysis, or conjugation in vivo, as well as oxidation, reduction, or hydrolysis in vivo. Also included are compounds that produce γ-oryzanol and its salts upon metabolism such as degradation.

  γ-Oryzanol and its salts are all isomers that can occur in the structure of the compound (for example, geometric isomers, optical isomers based on asymmetric carbon, rotational isomers, stereoisomers, tautomers, etc.) and A mixture of two or more of these isomers is also included and is not limited to the description of the structural formula for convenience.

  γ-Oryzanol and its salt may exist in the form of a hydrate depending on the kind thereof, and in the present invention, the hydrate is also included in γ-oryzanol or a salt thereof.

  In the IgE scavenger of the present invention, the content ratio of γ-oryzanol as an active ingredient, or a salt thereof, or a hydrate thereof is not limited and can be appropriately set. It is more preferable that oryzanol, or a salt thereof, or a hydrate thereof is purified to have a higher purity.

(2) Other components The IgE scavenger of the present invention can contain other components in addition to the above-mentioned active components. Examples of the other components include components that are required (or preferably used) according to various usages (usage forms) described later. These other components can be contained within a range that does not impair the IgE scavenging action exerted by the above-described active ingredients.

(3) Usage, Dosage Administration of the IgE scavenger of the present invention can be carried out by a known usage such as parenteral or oral, and is not limited, but preferably parenteral administration, particularly preferably transdermal administration. It is.

  Formulations (oral preparations, parenteral preparations, etc.) used in these various usages are excipients, fillers, fillers, binders, wetting agents, disintegrating agents, lubricants, surfactants, dispersions generally used in pharmaceutical production. An agent, a buffer, a preservative, a solubilizer, a preservative, a flavoring agent, a soothing agent, a stabilizer, an isotonic agent and the like can be appropriately selected and used, and can be prepared by a conventional method.

  The dose of the IgE scavenger of the present invention is determined in consideration of the proportion of the active ingredient in the preparation, and the administration subject (patient) age, weight, disease type / progression, administration route, administration frequency, administration A wide range can be set as appropriate in consideration of the period and the like.

  The case where the IgE scavenger of the present invention is used as a parenteral or oral preparation will be specifically described below.

  When used as a parenteral preparation, the form is not limited and includes, for example, intravenous injection (including infusion), intramuscular injection, intraperitoneal injection, subcutaneous injection, nasal spray, suppository, ointment, cream and Any of coating liquids and the like may be used, but among them, those intended for transdermal administration such as ointments, creams and coating liquids are preferable, and ointments are particularly preferable. In the case of various injections, for example, it can be provided in the state of a unit dose ampoule or a multi-dose container, or in the form of a lyophilized powder that is re-dissolved in a solution at the time of use. In addition to the above-mentioned active ingredient, the parenteral preparation can contain various known shaping materials and additives according to various forms as long as the effects of the active ingredient are not impaired. For example, in the case of various injections, water, glycerol, propylene glycol, aliphatic polyalcohols such as polyethylene glycol, and the like can be mentioned.

  The dose (per day) of the parenteral agent is not limited, and can be appropriately set so that an effective tissue concentration is maintained.

  When used as an oral preparation, its form is not limited, and for example, it may be any of tablets, capsules, granules, powders, pills, troches, liquids for internal use, suspensions, emulsions, syrups, etc. Alternatively, it may be a dry product which is redissolved when used. In addition to the above-mentioned active ingredient, the oral preparation can contain various types of excipients and additives according to various forms as long as the effect of the active ingredient is not impaired. For example, binder (syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, etc.), filler (lactose, sugar, corn starch, potato starch, calcium phosphate, sorbitol, glycine, etc.), lubricant (magnesium stearate, talc, Polyethylene glycol, silica, etc.), disintegrating agents (various starches, etc.), and wetting agents (sodium lauryl sulfate, etc.).

  The dose (per day) of the oral preparation is not limited, and can be appropriately set so that an effective blood concentration is maintained.

The blending ratio of the active ingredient in the oral preparation is not limited and can be appropriately set in consideration of the number of administrations per day.

3. Composition As described above, the antiallergic pharmaceutical composition, cosmetic composition, food composition, beverage composition and feed composition according to the present invention are all γ-oryzanol or pharmacologically. It is characterized by containing an acceptable salt or a hydrate thereof.

  In the present invention, γ-oryzanol or a pharmacologically acceptable salt thereof for the production of an antiallergic pharmaceutical composition, cosmetic composition, food composition, beverage composition and feed composition, Or the use of hydrates thereof.

  For the explanation of γ-oryzanol, which is an active ingredient in each of the above compositions, or a pharmacologically acceptable salt thereof, or a hydrate thereof, and other ingredients, the above 2. (1), (2 The description in) is equally applicable.

(1) Pharmaceutical composition The anti-allergic pharmaceutical composition of the present invention can be administered by a known method such as parenteral or oral, and is not limited, but is preferably parenteral.

  Formulations (oral preparations, parenteral preparations, etc.) used in these various usages are excipients, fillers, fillers, binders, wetting agents, disintegrating agents, lubricants, surfactants, dispersions generally used in pharmaceutical production. An agent, a buffer, a preservative, a solubilizer, a preservative, a flavoring agent, a soothing agent, a stabilizer, an isotonic agent and the like can be appropriately selected and used, and can be prepared by a conventional method.

  The dosage of the anti-allergic pharmaceutical composition of the present invention is determined in consideration of the mixing ratio of the active ingredient in the preparation, and the age, weight, type of disease, progress of administration, administration route, administration In consideration of the number of times, administration period, etc., it can be appropriately set over a wide range.

  The description in 2. (3) above can be similarly applied to the description of the case where the antiallergic pharmaceutical composition of the present invention is used as a parenteral or oral preparation.

  The present invention also relates to a method for treating and / or preventing allergic diseases, which comprises administering an effective amount of γ-oryzanol, or a pharmacologically acceptable salt thereof, or a hydrate thereof to a patient. Is also included. Examples of allergic diseases include atopic dermatitis, asthma, allergic rhinitis, and allergic enteritis.

(2) Cosmetic composition (cosmetics)
In the cosmetic composition of the present invention, the content of γ-oryzanol as an active ingredient, or a pharmacologically acceptable salt thereof, or a hydrate thereof is not limited and depends on the type of various cosmetics. And can be set as appropriate.

  Moreover, the method for obtaining the cosmetic composition of the present invention is not limited, and any known method and timing may be used to contain the active ingredient in a known production method according to the type of various cosmetics.

  Examples of the cosmetic composition of the present invention include known cosmetics such as lotions, emulsions, foundations, and lipsticks.

  The cosmetic product of the present invention allows the consumer to use it without worrying about the fear of various symptoms due to allergic reaction or reducing the fear.

(3) Food composition (food)
In the food composition of the present invention, the content ratio of γ-oryzanol as an active ingredient, or a pharmacologically acceptable salt thereof, or a hydrate thereof is not limited and depends on the type of each food. Can be set as appropriate.

  Moreover, the method for obtaining the food composition of the present invention is not limited, and the active ingredient may be contained in any known method and timing in a known production method according to the type of various foods.

  Examples of the food composition of the present invention include wheat flour-based foods, rice flour-based foods, and other foods.

  Examples of foods using flour include breads such as bread and confectionery bread; confectionery such as cookies and hot cakes; noodles such as udon, soba, Chinese soba and pasta.

  Examples of rice flour-based foods include rice cakes, Japanese sweets, and rice crackers.

  Examples of other foods include almost all known foods such as yogurt, ice cream and pudding.

  With the food of the present invention, consumers can eat without worrying about the fear of various symptoms due to allergic reaction or reducing the fear.

(4) Beverage composition (beverage)
In the beverage composition of the present invention, the content ratio of γ-oryzanol as an active ingredient, or a pharmacologically acceptable salt thereof, or a hydrate thereof is not limited, depending on the type of various beverages Can be set as appropriate.

  Moreover, the method of obtaining the drink composition of this invention is not limited, What is necessary is just to make it contain the said active ingredient by arbitrary methods and timings in the well-known manufacturing method according to the kind of various drinks.

  Examples of the beverage composition of the present invention include soft drinks, vegetable juice, fruit juice, tea, soup, mineral water, and the like, coffee, milk drinks, soy milk, and nourishing tonic drinks.

With the beverage of the present invention, consumers can drink without worrying about the fear of various symptoms due to allergic reaction or reducing the fear.
(5) Feed composition In the feed composition of the present invention, the content ratio of γ-oryzanol as an active ingredient, or a pharmacologically acceptable salt thereof, or a hydrate thereof is not limited. It can set suitably according to the kind of feed.

  Moreover, the method for obtaining the feed composition of the present invention is not limited, and the active ingredient may be contained in any known method and timing in a known production method according to the type of various feeds.

  Examples of the feed composition of the present invention include feeds given to various domestic animals such as cattle, horses, pigs, chickens, dogs, and cats or pet animals, and pellets added as additives or supplements to the feed Moreover, the thing of liquid form etc. are mentioned.

With the feed composition of the present invention, livestock is ingested with the expectation of reducing various symptoms due to various allergic reactions such as atopic dermatitis, allergic rhinitis, allergic enteritis, asthma, or allergy. be able to.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

1. Materials and methods
(1) Purification method of γ-oryzanol To 200 g of rice bran, 1 L of special grade hexane was added and stirred uniformly with a cup-type homogenizer at 12,000 rpm for 5 minutes, followed by suction filtration with No. 1 filter paper. After repeating this operation again, the obtained defatted soot was removed with hexane by an evaporator.

  Two-fold amount of distilled water was added to the resulting defatted soot, and the mixture was stirred uniformly for 12,000 revolutions for 5 minutes with a cup-type homogenizer, and then the soot was collected as a precipitate by centrifugation at 5,000 G for 20 minutes. Again, water-soluble components were extracted with distilled water, and after centrifugation, the resulting residue was dried by being left at an evaporator and at room temperature.

  Add 1 L of chloroform / methanol mixed solvent (mixing ratio 1: 1) to the resulting residue, stir uniformly with a cup-type homogenizer at 12,000 rpm for 5 minutes, and then suction filter with No. 1 filter paper. It was. After repeating this operation again, the obtained filtrate was dried with an evaporator to obtain purified γ-oryzanol.

(2) PCA reaction Oka et al. (OKA, T. et al., Microtubule disruption suppresses allergic response through the inhibition of calcium influx in the mast cell degranulation pathway., J. Immunol., 174, 4584-9, 2005) Was used to perform passive skin anaphylaxis (PCA) reaction as an allergy model. Eight week old male Sprague-Dawley rats (Charles River) weighing about 300 g were used. Anti-DNP-IgE (200 ng / ml: Sigma) was administered to the dorsal skin by intradermal injection under anesthesia. Two hours later, 1% Evans Blue (Sigma) and antigen (1 mg / ml DNP-human serum albumin (DNP-HSA)) added to 1 ml of saline were injected from the tail vein (iv). After 30 minutes, the skin of the rat was peeled, turned over and photographed. Next, the same round skin (about 1 cm ² ) was cut out, and Evans Blue that had exuded outside the blood vessel was extracted by incubating in 99% N, N-dimethylformamide (Wako Pure Chemical Industries) at 55 ° C. for 24 hours. . The supernatant was collected by centrifugation, and the OD at 650 nm was measured with a multi-label counter (Perkin Elmer). The leaching rate (%) was calculated with 0 to 200 ng / ml administered as 0 to 100%, respectively. Animals were handled and treated in accordance with the University of Tokyo facility guidelines.

(3) β-hexosaminidase degranulation The release of β-hexosaminidase was performed using the method of Oka et al. (J. Immunol., 174, 4584-9, 2005 (supra)). It was measured as an index of granule reaction. RBL-2H3 cells (American Type Culture Collection) were cultured at 37 ° C. in 24-well plates with gentle agitation. The supernatant enzyme and 0.5% triton X-100-releasing enzyme are incubated with p-nitrophenyl-N-acetyl-β-D-glucosamide (Sigma) in 0.04M sodium citrate and adjusted to pH 10 with NaOH. The reaction was stopped with adjusted 0.2M glycine. The OD at 405 nm was measured with a multi-label counter (Perkin Elmer). The degranulation rate (%) was calculated using the following formula.

Degranulation rate (%) = [OD of supernatant / (OD of supernatant + OD of triton X-100)] × 100
(4) Microscope observation RBL-2H3 cells on a cover glass were fixed with 3.7% formaldehyde at 37 ° C. for 10 minutes, and then washed with PBS. The cells were observed with a light microscope (Nikon).

(5) ELISA
The concentration of IgE was measured by a conventional method (BD biosciences). A 96-well plate was coated with anti-mouse IgE capture monoclonal antibody (PharMingen) and blocked with PBS containing 1% BSA. The purified standard anti-TNP-IgE (PharMingen) was diluted two-fold or incubated with γ-oryzanol or cycloartenyl ferulate (CAF) (Wako Pure Chemicals). Plates were incubated with IgE followed by biotinylated anti-mouse IgE (Serotec). Plates were washed and incubated with SAv-HRP, followed by incubation with 3,3 ′, 5,5′-tetramethylbenzidine (Sigma). The color reaction was stopped by the addition of 1M sulfuric acid. The OD at 405 nm was measured with a multi-label counter (Perkin Elmer).

(6) SDS-PAGE
SDS-PAGE was performed to detect the light and heavy chains of anti-DNP-IgE (Sigma) and anti-TNP-IgE (PharMingen). IgE after centrifugation (20,000 G, 4 ° C., 10 minutes) was treated by the usual method of SDS-PAGE. IgE light and heavy chains were separated by 10% SDS-PAGE and detected by silver staining (Silver staining II kit, Wako Pure Chemical Industries). The stained gel was imaged with a digital camera and stored, and the amount (intensity) of the protein was quantified using an image-processing program (Scion image). The concentration (density) (%) of the band was calculated with the control as 100%.

(7) Other chemicals We used cycloartenyl ferulate (CAF) (Wako Pure Chemicals), cremophor EL (Nacalai Tesque), and papain (Sigma).

2. result
(1) γ-Oryzanol which is the main component of cycloartenyl ferulate (CAF) First, the present inventor attempted to identify chemical components contained in γ-oryzanol derived from rice. As shown in A of FIG. 1, it was found that there are at least four types of ferulic acid esters, and CAF (see B of FIG. 1) is the main component of γ-oryzanol.

(2) Inhibition of mast cell degranulation reaction by cycloartenyl ferulate (CAF) First, the present inventors confirmed the effects of γ-oryzanol and CAF on the PCA reaction in vivo. Anti-DNP-IgE was injected intradermally followed by intravenous injection of DNP-HSA, which induces Evans Blue leaching. After anti-DNP-IgE was incubated with γ-oryzanol and CAF for 60 minutes, a mixed solution of anti-DNP-IgE was injected intradermally into the dorsal skin of rats. Evans blue leaching was inhibited in a concentration-dependent manner by γ-oryzanol (see A in FIG. 2) and CAF (see B in FIG. 2).

  Next, the present inventor confirmed the effect of CAF on mast cell degranulation in vitro. RBL-2H3 cells sensitized with anti-DNP-IgE released β-hexosaminidase after DNP-HSA stimulation. When anti-DNP-IgE incubated with CAF was added to RBL-2H3 cells, the degranulation reaction was inhibited by CAF in a concentration-dependent manner after DNP-HSA stimulation (see FIG. 3A). The inhibitory effect was dependent on the incubation time of CAF and anti-DNP-IgE (see FIG. 3B). The inventor also confirmed the cell shape after antigen stimulation. In RBL-2H3 cells not sensitized with anti-DNP-IgE, the cell morphology was not changed by DNP-HSA stimulation (see C in FIG. 3). On the other hand, RBL-2H3 cells sensitized with anti-DNP-IgE undergoes morphological changes by DNP-HSA stimulation (see D in FIG. 3). However, when RBL-2H3 cells were treated with anti-DNP-IgE incubated with CAF, the cell shape was not changed by subsequent DNP-HSA stimulation (see E in FIG. 3).

  The present inventor examined whether CAF could inhibit the degranulation reaction of RBL-2H3 cells already sensitized with anti-DNP-IgE. As a result, CAF could not suppress the degranulation reaction induced by DNP-HSA in RBL-2H3 cells previously sensitized with anti-DNP-IgE (see FIG. 4).

(3) IgE capture by cycloartenyl ferulate (CAF) From the above findings, it is considered that γ-oryzanol and CAF have some influence on the ability of IgE to bind to the FcεRI receptor. In order to clarify this point, the present inventor first measured the influence of CAF on the IgE concentration by ELISA. When anti-TNP-IgE was incubated with γ-oryzanol or CAF for 60 minutes, the IgE concentration in the solution detectable by ELISA decreased depending on the concentration of γ-oryzanol or CAF (see FIGS. 5A to C). . The effect of CAF on the IgE concentration also depended on the incubation time (see D in FIG. 5). From these results, it is considered that anti-IgE antibody in ELISA cannot detect IgE after incubation with CAF.

  There seem to be at least two possibilities for the effect of CAF on IgE. One possibility is the capture (sequestration) of IgE from anti-IgE antibodies, and another possibility is a conformational change of IgE. To investigate this point, the IgE molecule was analyzed by SDS-PAGE. High and low molecular bands were detected by silver staining (see Fig. 6). The upper band (not confirmed after treatment with papain) is the heavy chain of IgE, and the lower band is the light chain of IgE (see FIGS. 6A and 6D). Incubating IgE with CAF had no effect on either the electrophoretic mobility or amount of IgE molecules (see the third band or graph from the left of B, C, E, and F in Figure 6). reference). When IgE was incubated with CAF, the amount of IgE in the supernatant after centrifugation decreased (see the fourth band or graph from the left of B, C, E and F in FIG. 6). From these results, it is considered that CAF captured IgE and the complex of CAF and IgE was precipitated by centrifugation.

Since CAF has a hydrophobic region, the present inventor considered that a hydrophobic interaction between the two is necessary for the binding of CAF and IgE. Therefore, the present inventor confirmed the influence of the surfactant on the effect of CAF on IgE. Anti-DNP-IgE incubated with γ-oryzanol and anti-DNP-IgE incubated with CAF in the presence of a low cytotoxic surfactant (cremophor EL), both of Evans Blue in the PCA reaction The exudation outside the blood vessels (see FIG. 7A) and the degranulation reaction (see FIG. 7B) could not be inhibited. From these results, it is considered that the hydrophobicity of CAF is necessary for capturing IgE.

3. Discussion In this example, the present inventor confirmed the effect of PCA reaction on γ-oryzanol extracted from rice bran (see FIG. 2). As a result, as shown in FIG. 2A, it was found that γ-oryzanol significantly inhibited the reaction. Since cycloartenyl ferulate (CAF) is one of the main components of γ-oryzanol (see FIG. 1), the present inventors next tested the effect of CAF on the PCA reaction. As shown in FIG. 2B, CAF inhibited the PCA reaction.

  In order to confirm that the degranulation reaction of mast cells is inhibited by this inhibitory effect of CAF, the present inventors further confirmed the effect of CAF in the degranulation reaction using RBL-2H3 cells (see FIG. 3). ). As a result, as shown in FIG. 3A, CAF inhibited mast cell degranulation in a concentration-dependent manner. This inhibitory effect was dependent on the incubation time of CAF and anti-DNP-IgE, as shown in FIG. 3B. Furthermore, once IgE bound to mast cells, CAF could not inhibit the degranulation reaction (see FIG. 4). These results indicate that CAF has some effect on the ability of IgE to bind to the FcεRI receptor.

  In order to clarify how CAF inhibits the binding of IgE, the present inventor first measured the concentration of IgE by ELISA. As shown in FIG. 5, γ-oryzanol and CAF decreased the concentration of IgE detected by the anti-IgE antibody. However, in the analysis by SDS-PAGE, IgE was still present in the solution even after incubation with CAF (see the third band from the left of B and E in FIG. 6). On the other hand, IgE incubated with CAF was removed from the supernatant by centrifugation (see the fourth band from the left of B, C, E and F in FIG. 6 or the graph). Therefore, the present inventor considered that the anti-IgE antibody in ELISA could not be detected because CAF captured IgE. Considering that IgE is removed from the supernatant by centrifugation, CAF is considered to form a large cluster.

  Furthermore, since γ-oryzanol and CAF could not inhibit allergic reaction in the presence of cremophor EL (see A and B in Fig. 7), hydrophobic interaction between CAF and IgE was necessary to inhibit allergic reaction. The action is considered necessary.

  Since CAF is an ester of ferulic acid and it has already been reported that ferulic acid has a variety of pharmacological activities, the present inventors have tested the effect of ferulic acid alone on allergic reactions. However, ferulic acid (less than 30 μM) failed to attenuate the PCA reaction and did not inhibit the release of β-hexosaminidase. From these results, it is considered that the hydrophobicity of CAF is necessary for exerting the IgE capturing action.

  As mentioned earlier, the further importance of IgE in allergic diseases is still drawing attention today. Thus, IgE is a key target in allergic diseases. The present inventor has demonstrated that CAF in γ-oryzanol extracted from rice bran captures IgE and inhibits binding to FcεRI receptor, resulting in reduced allergic reaction. Such a natural product contained in rice bran can be used as a therapeutic agent for allergic diseases, and can also be used for prevention, for example, functional foods.

It is a figure which shows that the ferulic acid cycloartenyl (CAF) is one of the main components of (gamma)-oryzanol extracted from the rice bran.

A is the ratio of ferulic acid ester in rice bran, and B is the structure of CAF.
It is a figure which shows that gamma-oryzanol and CAF weaken PCA reaction.

Anti-DNP-IgE (200 ng / ml) was incubated with the indicated concentrations of γ-oryzanol (A) or CAF (B) for 60 minutes and injected into rat dorsal skin. The upper panel shows a typical picture of the PCA reaction. The lower graph shows analytical data on Evans Blue that has migrated (number of test cases: 6 to 8 cases).
It is a figure which shows that CAF inhibits the degranulation reaction of RBL-2H3 cell.

  Anti-DNP-IgE (50 ng / ml) was incubated with the indicated concentration of CAF for 60 minutes (A) or incubated with 10 μM CAF for the indicated time (B). RBL-2H3 cells were incubated with IgE and CAF for 15 minutes and stimulated with 10 ng / ml DNP-HSA for 15 minutes. The percent release of β-hexosaminidase by DNP-HSA was calculated and the results were expressed as the mean ± SE of 6 trials.

RBL-2H3 cells were not sensitized (C), sensitized with IgE for 15 minutes (D), or sensitized with IgE and 10 μM CAF for 15 minutes (E). Subsequently, it stimulated with 10 ng / ml DNP-HSA for 15 minutes. Cells were fixed with formaldehyde and observed under a microscope. Results are representative of 4 trials.
It is a figure which shows that CAF cannot inhibit the degranulation reaction of IgE binding RBL-2H3 cells.

RBL-2H3 cells were not sensitized (-IgE) or sensitized with 50 ng / ml anti-DNP-IgE for 15 minutes. Subsequently, it was incubated for 60 minutes in the presence or absence of 30 μM CAF. Cells were then stimulated with 10 ng / ml DNP-HSA for 15 minutes. The percent release of β-hexosaminidase by DNP-HSA was calculated and the results were expressed as the mean ± SE of 8 trials.
FIG. 6 shows that γ-oryzanol and CAF decrease the IgE concentration detected by ELISA.

Anti-TNP-IgE (200 ng / ml for A and B, 50 ng / ml for C) was incubated with the indicated concentrations of γ-oryzanol (A) or CAF (B and C) for 60 minutes. Anti-TNP-IgE (200 ng / ml) was incubated with 10 μM CAF for the indicated times (D). IgE concentrations were measured by ELISA and the results were expressed as the mean ± SE of 4 studies.
It is a figure which shows that CAF captures IgE.

After centrifugation, anti-DNP-IgE (A to C; 10 μg / ml) or anti-TNP-IgE (D to F; 10 μg / ml) was added in the absence (control) or presence (A and A) of 2 μM papain. D) for 60 minutes or with 30 μM CAF for 60 minutes (B and E). The solution was then centrifuged again (B and E; 20,000 G, 4 ° C., 10 minutes) or not centrifuged. The heavy chain (HC) and light chain (LC) band densities in B and E were calculated and are shown in C and F, respectively.
It is a figure which shows that CAF cannot inhibit an allergic reaction in presence of cremophor EL.

  Incubate anti-DNP-IgE (200 ng / ml) in the presence of 0.01% cremophor EL in the absence or presence of 18 μg / ml γ-oryzanol (γ-ory) or 30 μM CAF Incubated for 60 minutes in the presence of. Thereafter, it was injected into the dorsal skin of the rat. The upper panel shows a typical picture of the PCA reaction. The graph below shows analytical data on Evans Blue that has migrated (number of test cases: 4).

Is anti-DNP-IgE (50 ng / ml) incubated in the presence or absence of 0.01% cremophor EL for 60 minutes in the absence or presence of 18 μg / ml γ-oryzanol (γ-ory)? Or 60 min in the presence of 30 μM CAF. These sensitized RBL-2H3 cells for 15 minutes. The% release of β-hexosaminidase by DNP-HSA was calculated and the results were expressed as the mean ± SE of 4-12 trials.

Claims (2)

  Including cycloartenyl ferulate, or a pharmacologically acceptable salt thereof, or a hydrate thereof (however, components other than cycloartenyl ferulate among components contained in γ-oryzanol, or pharmacology thereof) Free of chemically acceptable salts or hydrates thereof), IgE scavengers.   The IgE scavenger according to claim 1, which does not contain a surfactant.